Dog melanin-concentrating hormone receptor

ABSTRACT

The present invention features polypeptides and nucleic acids related to a dog MCH receptor and uses of such polypeptides and nucleic acids. The dog MCH receptor is a G protein coupled receptor whose activity is stimulated by MCH binding.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to provisional application U.S.Ser. No. 60/219,669, filed Jul. 21, 2000, hereby incorporated byreference herein.

BACKGROUND OF THE INVENTION

The references cited herein are not admitted to be prior art to theclaimed invention.

Neuropeptides present in the hypothalamus play a major role in mediatingthe control of body weight. (Flier et al., 1998, Cell, 92, 437–440.)Melanin-concentrating hormone (MCH) is a cyclic 19-amino acidneuropeptide synthesized as part of a larger pre-prohormone precursor inthe hypothalamus that also encodes neuropeptides NEI and NGE. (Nahon etal., 1990, Mol. Endocrinol., 4, 632–637.) MCH was first identified insalmon pituitary, and in fish MCH affects melanin aggregation thusaffecting skin pigmentation. In trout and in eels MCH has also beenshown to be involved in stress induced or CRF-stimulated ACTH release.(Kawauchi et al., 1983, Nature, 305, 321–323.)

In humans two genes encoding MCH have been identified that are expressedin the brain. (Breton et al., 1993, Mol. Brain Res., 18, 297–310.) Inmammals MCH has been localized primarily to neuronal cell bodies of thehypothalamus which are implicated in the control of food intake,including perikarya of the lateral hypothalamus and zona inertia.(Knigge et al., 1996, Peptides, 17, 1063–1073.)

Pharmacological and genetic evidence suggest that the primary mode ofMCH action is to promote feeding (orexigenic). MCH mRNA is up regulatedin fasted mice and rats and in the ob/ob mouse. (Qu et al., 1996,Nature, 380, 243–247.) Injection of MCH centrally (ICV) stimulates foodintake and MCH antagonizes the hypophagic effects seen with α-melanocytestimulating hormone (αMSH). (Qu et al., 1996, Nature, 380, 243–247.)MCH-deficient mice are lean, hypophagic, and have increased metabolicrate. (Shimada et al., 1998, Nature, 396, 670–673.)

MCH action is not limited to modulation of food intake as effects on thehypothalamic-pituitary-axis have been reported. (Nahon, 1994, CriticalRev. in Neurobiol., 8, 221–262.) MCH may be involved in the bodyresponse to stress as MCH can modulate the stress-induced release of CRFfrom the hypothalamus and ACTH from the pituitary. In addition, MCHneuronal systems may be involved in reproductive or maternal function.

Several references describe a receptor that is indicated to bind MCH.(Chambers et al., 1999, Nature, 400, 261–265; Saito et al., 1999,Nature, 400, 265–269; Bächner et al., 1999, FEBS Letters, 457, 522–524;Shimomura et al., 1999, Biochemical and Biophysical ResearchCommunications, 261, 622–626; and Lembo et al., 1999, Nat. Cell Biol.,1, 267–271.)

SUMMARY OF THE INVENTION

The present invention features polypeptides and nucleic acids related toa dog MCH receptor and uses of such polypeptides and nucleic acids. Thedog MCH receptor is a G protein coupled receptor whose activity isstimulated by MCH binding.

Polypeptides related to a dog MCH receptor contain a region of at least9 contiguous amino acids that is present in a dog MCH receptor. Suchpolypeptides may contain additional regions including regions present,or not present, in a dog MCH receptor.

Nucleic acids related to a dog MCH receptor contain a region of at least18 contiguous nucleotides that is present in a dog MCH receptor nucleicacid. Such nucleic acids may contain additional regions includingregions present, or not present, in a dog MCH receptor nucleic acid.

Thus, a first aspect of the present invention describes a purifiedpolypeptide comprising a unique amino acid region of a dog MCH receptor.The unique region is at least 9 amino acids in length.

A “unique amino acid region” of a dog MCH receptor is a region ofcontiguous amino acids present in SEQ. ID. NO. 1 that is not present inSEQ. ID. NOs. 2 or 3. SEQ. ID. NO. 1, which is referred to herein as adog MCH receptor, was derived from dog nucleic acid using a human primerand may thus contain one or more N-terminal amino acids corresponding toa human source rather than a dog source. SEQ. ID. NO. 2 is a human MCHreceptor amino acid sequence and SEQ. ID. NO. 3 is a rat MCH receptoramino acid sequence. The unique region may contain segments ofcontiguous amino acids present in SEQ. ID. NOs. 2 or 3 smaller than theindicated unique region size.

A “purified polypeptide” represents at least 10% of the total proteinpresent in a sample or preparation. In preferred embodiments, thepurified polypeptide represents at least about 50%, at least about 75%,or at least about 95% of the total protein in a sample or preparation.Reference to “purified polypeptide” does not require that thepolypeptide has undergone any purification and may include, for example,chemically synthesized polypeptide that has not been purified.

Another aspect of the present invention describes a purified nucleicacid comprising a nucleotide sequence encoding for a unique amino acidregion from a dog MCH receptor. The encoded for region is at least 9amino acids in length.

A “purified nucleic acid” represents at least 10% of the total nucleicacid present in a sample or preparation. In preferred embodiments, thepurified nucleic acid represents at least about 50%, at least about 75%,or at least about 95% of the total nucleic acid in a sample orpreparation. Reference to “purified nucleic acid” does not require thatthe nucleic acid has undergone any purification and may include, forexample, chemically synthesized nucleic acid that has not been purified.

Another aspect of the present invention describes a purified nucleicacid comprising a unique nucleotide sequence region of a dog MCHreceptor nucleic acid sequence, or the complement thereof. The uniquenucleotide sequence region is at least 18 nucleotides in length.

A “unique nucleotide sequence region” of a dog MCH receptor nucleic acidis a region that comprises at least 18 contiguous nucleotides of SEQ.ID. NO. 4 that is not present in SEQ. ID. NOs. 5 or 6. SEQ. ID. NO. 4,which is referred to herein as a nucleotide sequence encoding for a dogMCH receptor, was derived from dog nucleic acid using a human primer andmay thus contain one or more 5′ nucleotides corresponding to a humansource rather than a dog source. SEQ. ID. NO. 5 is the nucleotidesequence encoding for a human MCH receptor and SEQ. ID. NO. 6 is thenucleotide sequence encoding for a rat MCH receptor. The unique regionmay contain segments of contiguous nucleotides present in SEQ. ID. NOs.5 or 6 smaller than the indicated unique region size.

Another aspect of the present invention describes a nucleic acidcomprising a recombinant nucleotide sequence encoding for a unique aminoacid region of a dog MCH receptor. In different embodiments the nucleicacid is an expression vector or is part of a host genome.

A “recombinant nucleotide sequence” is a sequence that is present on anucleic acid containing one or more nucleic acid regions not naturallyassociated with that sequence. Examples of such regions that may bepresent with the sequence include one or more regulatory elements notnaturally associated with the sequence, viral elements, and selectablemarkers.

Another aspect of the present invention describes a recombinant cellcomprising an expression vector encoding for a unique amino acid regionof a dog MCH receptor. The expression vector contains a promoter that isfunctionally coupled to nucleic acid encoding for the unique region andis recognized by an RNA polymerase present in the cell.

Another aspect of the present invention describes a recombinant cellmade by introducing an expression vector encoding for a unique aminoacid region of a dog MCH receptor into a cell. The expression vector canbe used to insert the dog nucleic acid into the genome of the host, orcan exist as an autonomous piece of nucleic acid.

Another aspect of the present invention describes a method of measuringthe ability of a test compound to affect MCH receptor activity. Themethod involves providing the compound to a recombinant cell expressinga functional MCH receptor containing a unique dog amino acid region froma recombinant nucleic acid and measuring MCH receptor activity.Preferably, the recombinant nucleic acid is present on an expressionvector.

Another aspect of the present invention describes a method of producinga MCH receptor polypeptide. The method involves the step of growing arecombinant cell able to express a dog MCH receptor polypeptide underconditions wherein the polypeptide is expressed from an expressionvector.

Other features and advantages of the present invention are apparent fromthe additional descriptions provided herein including the differentexamples. The provided examples illustrate different components andmethodology useful in practicing the present invention. The examples donot limit the claimed invention. Based on the present disclosure theskilled artisan can identify and employ other components and methodologyuseful for practicing the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a comparison of the amino acid sequence for a dog MCHreceptor (SEQ. ID. NO. 1), a human MCH receptor (SEQ. ID. NO. 2), and arat MCH receptor (SEQ. ID. NO. 3).

FIGS. 2A–2C illustrate a comparison of the nucleotide sequence encodingfor a dog MCH receptor (SEQ. ID. NO. 4), a human MCH receptor (SEQ. ID.NO. 5), and a rat MCH receptor (SEQ. ID. NO. 6).

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides and nucleic acids related to a dog MCH receptor arepreferably used in an in vitro functional assay measuring whether acompound acts differently at the dog receptor than at the humanreceptor, or affects MCH receptor activity. Such assays can be used tohelp evaluate whether a dog model provides a useful test system inlooking for a human therapeutic compound and for assaying for compoundsactive at the MCH receptor.

The MCH receptor provides a target to achieve different beneficialeffects in a patient. Preferably, MCH receptor activity is modulated toachieve one or more of the following: weight loss, weight gain, treatcancer (e.g., colon or breast), reduce pain, treat diabetes, reducestress, or teat sexual dysfunction.

Modulation of MCH receptor activity can be achieved by evoking aresponse at the MCH receptor or by altering a response evoked by an MCHreceptor agonist or antagonist. Compounds modulating MCH receptoractivity include agonists, antagonists, and allosteric modulators.Generally, MCH receptor antagonists and allosteric modulators negativelyaffecting activity will be used to achieve weight loss, treat cancer(e.g., colon or breast), reduce pain, reduce stress, and/or teat sexualdysfunction; and MCH receptor agonists and allosteric modulatorspositively affecting activity will be used to produce a weight gain.

Preferably, MCH receptor activity is modulated to achieve a weight lossor to treat diabetes in a patient. Diabetes mellitus can be treated bymodulating MCH receptor activity to achieve, for example, one or both ofthe following: enhancing glucose tolerance or decreasing insulinresistance.

Excessive body weight is a contributing factor to different diseases,including hypertension, diabetes, dyslipidemias, cardiovascular disease,gall stones, osteoarthritis, and certain forms of cancers. Bringingabout a weight loss can be used, for example, to reduce the likelihoodof such diseases and as part of a treatment for such diseases. Weightreduction can be achieved by modulating MCH receptor activity to obtain,for example, one or more of the following effects: reducing appetite,increasing metabolic rate, reducing fat intake, or reducing carbohydratecraving.

Facilitating a weight gain, maintenance in weight, or appetite increaseis particularly useful for a patient having a disease or disorder, orunder going a treatment, accompanied by weight loss. Examples ofdiseases or disorders accompanied by weight loss include anorexia,bulimia, cancer cachexia, AIDS, wasting, cachexia, and wasting in frailelderly. Examples of treatments accompanied by weight loss includechemotherapy, radiation therapy, temporary or permanent immobilization,and dialysis.

MCH Receptor Related Polypeptides

Polypeptides related to the dog MCH receptor preferably contain a uniquedog amino acid region. In addition to the unique amino acid region,regions that may, or may not, be related to the dog MCH receptorpolypeptide may be present. Such polypeptides have a variety of uses,such as providing a component of a functional MCH receptor; being usedas an immunogen to produce antibodies binding to the MCH receptor; beingused as a target to identify compounds binding to the MCH receptor;and/or being used in assays measuring the ability of a compound toaffect MCH receptor activity.

Unique dog amino acid regions can readily be identified based on acomparison of the dog MCH receptor sequence described herein, with thehuman and rat MCH receptor amino acid sequences. Such a sequencecomparison is illustrated in FIG. 1. Examples of unique dog amino acidregions include the following:

LEASLLPPGP, SEQ. ID. NO. 7 SEGPDNLTSAGP, SEQ. ID. NO. 8 RRTGNVSYIN, SEQ.ID. NO. 9 PFPGGTVGCG, and SEQ. ID. NO. 10 ILQRMMSSVA. SEQ. ID. NO. 11

The definition of unique amino acid region is with respect to the humanand rat MCH receptors. Thus, a unique amino acid region may be presentin a MCH receptor amino acid sequence from one or more species otherthan the human or rat sequences, or in a non-MCH receptor sequence.

In different embodiments a dog MCH receptor related polypeptidecomprises or consists of a unique amino acid region at least 18, atleast 27, or at least 54, amino acids in length. Preferably, the dog MCHreceptor related polypeptide comprises or consists of the amino acidsequence of SEQ. ID. NO. 1.

Polypeptides can be produced using standard techniques including thoseinvolving chemical synthesis and those involving biochemical synthesis.Techniques for chemical synthesis of polypeptides are well known in theart. (See e.g., Vincent, in Peptide and Protein Drug Delivery, New York,N.Y., Dekker, 1990.)

Biochemical synthesis techniques for polypeptides are also well known inthe art. Such techniques employ a nucleic acid template for polypeptidesynthesis. The genetic code providing the sequences of nucleic acidtriplets coding for particular amino acids is well known in the art.(See, e.g., Lewin GENES IV, p. 119, Oxford University Press, 1990.)Examples of techniques for introducing nucleic acid into a cell andexpressing the nucleic acid to produce protein are provided inreferences such as Ausubel, Current Protocols in Molecular Biology, JohnWiley, 1987–1998, and Sambrook et al., in Molecular Cloning, ALaboratory Manual, 2^(nd) Edition, Cold Spring Harbor Laboratory Press,1989.

Functional MCH Receptor Derivatives

Functional MCH receptors are stimulated by MCH binding. Functional MCHreceptors include the MCH receptor of SEQ. ID. NO. 1, and receptorshaving MCH receptor activity and containing a unique dog amino acidregion as a component.

Starting with a MCH receptor obtained from a particular source,derivatives can be produced having MCH receptor activity. Suchderivatives include polypeptides with amino acid substitutions,additions and deletions. Changes made to produce functional derivativesshould be made outside of the MCH binding domain and in a manner notaltering the tertiary structure. The ability of a polypeptide to haveMCH receptor activity can be confirmed using techniques such as thosemeasuring G-protein activity.

The sequence comparison provided in FIG. 1 illustrates amino acids thatvary between the human, rat, and dog MCH receptor. Such variable aminoacids are good targets for alterations.

Additionally, amino acids are classified into certain types based on thestructure of their R-groups. Substituting different amino acids within aparticular group, such as substituting valine for leucine, arginine forlysine, and asparagine for glutamine may not cause a change infunctionality of the polypeptide.

MCH Antibodies

Antibodies recognizing a dog MCH receptor polypeptide can be producedusing a polypeptide of SEQ. ID. NO. 1 or a fragment thereof as animmunogen. Fragments should be at least 9 amino acids in length andpreferably contain a unique amino acid region.

Antibodies to the MCH receptor have different uses such as being used toidentify the presence of MCH receptor polypeptides and for isolating MCHreceptor polypeptides. Examples of techniques for producing and usingantibodies are described in Ausubel Current Protocols in MolecularBiology, John Wiley, 1987–1998, Harlow et al., Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory, 1988, and Kohler et al., 1975,Nature, 256, 495–497.

Binding Assays

Assays measuring the ability of a compound to bind the dog MCH receptorcan be performed using a polypeptide of SEQ. ID. NO. 1 or a fragmentthereof as a target. Fragments should be at least 9 amino acids inlength and contain a site to which either an agonist, antagonist, orallosteric modulator binds. Different types of assay formats can beemployed including competitive and non-competitive assays.

Compounds identified as binding to a full-length receptor or a receptorfragment can be used to determine the locus of a binding site by testingthe ability of the compound to bind to smaller length fragments. Forexample, MCH binds to the MCH receptor and labeled MCH can be used toidentify that portion of the receptor to which MCH binds. Fragmentsidentified as containing a compound binding site can be used to test foradditional compounds that bind to the binding site.

Preferred polypeptide fragments used in a binding assay consist of aunique amino acid region. However, fragments containing additional aminoacid sequences can be employed, for example, to facilitate attachment toa column.

Binding assays can be performed using individual compounds orpreparations containing different compounds. A preparation containingdifferent compounds wherein one or more compounds bind to the MCHreceptor can be divided into smaller groups to identify compound(s)binding to the MCH receptor. In an embodiment of the present invention atest preparation containing at least 10 compounds is used in a bindingassay.

Binding assays can be performed using recombinantly produced MCHreceptor polypeptides present in different environments. Suchenvironments include, for example, cell extracts and purified cellextracts containing the MCH receptor polypeptide expressed fromrecombinant nucleic acid; and the use of a purified MCH receptorpolypeptide produced by recombinant means which is introduced into adifferent environment.

Functional Assays

Assays involving functional dog MCH receptors and chimeric receptors canbe employed to select for compounds active at the MCH receptor and toevaluate the ability of a compound to affect receptor activity. MCHreceptor activity can be measured using different techniques such asdetecting a change in the intracellular conformation of the MCHreceptor, measuring G protein activity, or measuring the level ofintracellular messengers.

Recombinantly expressed MCH receptor polypeptides can be used tofacilitate determining whether a compound is active at the MCH receptoror another receptor. For example, the MCH receptor can be expressed byan expression vector in a cell line such as HEK 293, COS 7, and CHO notnormally expressing the receptor, wherein the same cell line without theexpression vector or with an expression vector not encoding a MCHreceptor can act as a control.

MCH receptor activity can be measured, for example, by assays measuringthe phospholipase C signal transduction pathway. Activity of thephospholipase C signal transduction pathway can be measured usingstandard techniques such as those measuring intracellular Ca²⁺. Examplesof techniques well known in the art that can be employed to measure Ca²⁺include the use of dyes such as Fura-2 and the use ofCa²⁺-bioluminescent sensitive reporter proteins such as aequorin. Anexample of a cell line employing aequorin to measure G protein activityis HEK293/aeq17. (Button et al., 1993, Cell Calcium, 14, 663–671, andFeighner et al., 1999, Science, 284, 2184–2188, both of which are herebyincorporated by reference herein.)

Chimeric receptors containing one or more MCH receptor regionsfunctionally coupled to polypeptides from other G proteins can also beused to measure activity. A chimeric MCH receptor contains an N-terminalextracellular domain; a transmembrane domain made up of transmembraneregions, extracellular loop regions, and intracellular loop regions; andan intracellular carboxy terminus domain. Preferred chimerics containone or more of these different domains from a dog MCH receptor.

The specificity of G protein coupling is determined by intracellulardomain(s). A chimeric G protein coupled receptor can be produced tofunctionally couple to a particular G protein. For example a G proteinthat normally couples to Gs can be coupled to Gq or Gi allowing for thedetection of receptor activity by measuring Gq or Gi activity.Techniques for producing chimeric receptors and measuring G proteincoupled responses are provided for in, for example, InternationalApplication No. WO 97/05252, and U.S. Pat. No. 5,264,565, both of whichare hereby incorporated by reference herein.

Functional assays can be performed using individual compounds orpreparations containing different compounds. A preparation containingdifferent compounds where one or more compounds affect MCH receptor orchimeric receptor activity can be divided into smaller groups ofcompounds to identify the compound(s) affecting MCH receptor activity.In an embodiment of the present invention a test preparation containingat least 10 compounds is used in a functional assay.

Functional assays can be performed using recombinantly produced MCHreceptor polypeptides or chimeric receptor polypeptides present indifferent environments. Such environments include, for example, cellextracts, and purified cell extracts, containing the MCH receptorpolypeptide expressed from recombinant nucleic acid; and the use of apurified MCH receptor polypeptide produced by recombinant means that isintroduced into a different environment.

MCH Receptor Related Nucleic Acid

Nucleic acids related to the dog MCH receptor nucleic acid preferablycontain a unique dog nucleotide sequence region or the complementthereof. Such nucleic acids have a variety of uses, such as being usedas a hybridization probe or PCR primer to identify the presence of dogMCH nucleic acid; being used as a hybridization probe or PCR primer toidentify or clone nucleic acid encoding for receptors related to the MCHreceptor from different sources; and/or being used for recombinantexpression of a dog MCH receptor polypeptide.

Unique dog nucleic acid regions can readily be identified based on acomparison of the dog MCH receptor nucleic acid sequences describedherein, with the human and the rat MCH receptor nucleic acid sequences.Such a sequence comparison is illustrated in FIGS. 2A–2C.

Examples of unique dog nucleic acid regions include the following:

SEQ. ID. NO. 12 CCTCGGAGGGCCCGGACAACC, SEQ. ID. NO. 13ACCTCTGCCGGGCCACCTCGT, SEQ. ID. NO. 14 GCCCTTCGTGGTCATCACAGCCGCGTAT,SEQ. ID. NO. 15 TGTCCTCGGTAGCCCCTGCCTCTCAA, and SEQ. ID. NO. 16TTCGAGCCGTCAGCAATGCT.

The guidance provided in the present application can be used to obtainthe nucleic acid sequence encoding the full-length dog MCH receptor, toobtain nucleic acids encoding for MCH receptors from additional sources,and to artificially produce a MCH receptor. Obtaining nucleic acidsencoding a MCH receptor from different sources is facilitated using setsof degenerative probes and primers and by the proper selection ofhybridization conditions. Sets of degenerative probes and primers areproduced taking into account the degeneracy of the genetic code.Adjusting hybridization conditions is useful for controlling probe orprimer specificity to allow for hybridization to nucleic acids havingsimilar sequences.

Techniques employed for hybridization detection and PCR cloning are wellknown in the art. Nucleic acid detection techniques are described, forexample, in Sambrook et al., in Molecular Cloning, A Laboratory Manual,2^(nd) Edition, Cold Spring Harbor Laboratory Press, 1989. PCR cloningtechniques are described, for example, in White, Methods in MolecularCloning, volume 67, Humana Press, 1997.

MCH receptor probes and primers can be used to screen nucleic acidlibraries containing, for example, genomic DNA or cDNA. Such librariesare commercially available, and can be produced using techniques such asthose described in Ausubel, Current Protocols in Molecular Biology, JohnWiley, 1987–1998.

Starting with a particular amino acid sequence and the known degeneracyof the genetic code, a large number of different encoding nucleic acidsequences can be obtained. The degeneracy of the genetic code arisesbecause almost all amino acids are encoded by different combinations ofnucleotide triplets or “codons”. The translation of a particular codoninto a particular amino acid is well known in the art (see, e.g., LewinGENES IV, p. 119, Oxford University Press, 1990). Amino acids areencoded by codons as follows:

-   A=Ala=Alanine: codons GCA, GCC, GCG, GCU-   C=Cys=Cysteine: codons UGC, UGU-   D=Asp=Aspartic acid: codons GAC, GAU-   E=Glu=Glutamic acid: codons GAA, GAG-   F=Phe=Phenylalanine: codons WJC, UUU-   G=Gly=Glycine: codons GGA, GGC, GGG, GGU-   H=His=listidine: codons CAC, CAU-   I=Ile=Isoleucine: codons AUA, AUC, AUU-   K=Lys=Lysine: codons AAA, AAG-   L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU-   M=Met=Methionine: codon AUG-   N=Asn=Asparagine: codons AAC, AAU-   P=Pro=Proline: codons CCA, CCC, CCG, CCU-   Q=Gln=Glutamine: codons CAA, CAG-   R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU-   S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU-   T=Thr=Threonine: codons ACA, ACC, ACG, ACU-   V=Val=Valine: codons GUA, GUC, GUG, GUU-   W=Trp=Tryptophan: codon UGG-   Y=Tyr=Tyrosine: codons UAC, UAU.

Nucleic acid having a desired sequence can be synthesized using chemicaland biochemical techniques. Examples of chemical techniques aredescribed in Ausubel, Current Protocols in Molecular Biology, JohnWiley, 1987–1998, and Sambrook et al., in Molecular Cloning, ALaboratory Manual, 2^(nd) Edition, Cold Spring Harbor Laboratory Press,1989.

Biochemical synthesis techniques involve the use of a nucleic acidtemplate and appropriate enzymes such as DNA and/or RNA polymerases.Examples of such techniques include in vitro amplification techniquessuch as PCR and transcription based amplification, and in vivo nucleicacid replication. Examples of suitable techniques are provided byAusubel, Current Protocols in Molecular Biology, John Wiley, 1987–1998,Sambrook et al., in Molecular Cloning, A Laboratory Manual, 2^(nd)Edition, Cold Spring Harbor Laboratory Press, 1989, and Kacian et al.,U.S. Pat. No. 5,480,784.

In different embodiments dog MCH receptor related nucleic acid comprisesor consists of a unique nucleic acid region, or the complement thereof,at least 27 or at least 54 bases in length. Preferably, the dog MCHreceptor related nucleic acid comprises or consists of the nucleic acidsequence of SEQ. ID. NO. 4.

MCH Receptor Probes

Detection probes for the dog MCH receptor preferably contain a uniquedog nucleic acid region, or the complement thereof. Such probes cancontain additional nucleic acid that may, or may not, be complementaryto dog MCH receptor nucleic acid. Preferably, additional nucleic acidthat is present has a particular purpose such as providing for increasedspecificity, being a reporter sequence, or being a capture sequence.However, additional nucleic acid need not have a particular purpose.

Probes for the MCH receptor can specifically hybridize to MCH receptortarget nucleic acid under appropriate hybridization conditions (i.e.,distinguish target nucleic acid from one or more non-target nucleic acidmolecules). A preferred non-target nucleic acid is either nucleic acidencoding for the human MCH receptor or the complement thereof.Hybridization occurs through complementary nucleotide bases present onthe probe and MCH receptor nucleic acid. Hybridization conditionsdetermine whether two molecules have sufficiently strong interactionswith each other to form a stable hybrid.

Probes are composed of nucleic acids or derivatives thereof such asmodified nucleic acid and peptide nucleic acid. Modified nucleic acidincludes nucleic acid with one or more altered sugar groups, alteredinternucleotide linkages, and/or altered nucleotide purine or pyrimidinebases. References describing modified nucleic acid include InternationalPublication No. WO 98/02582, U.S. Pat. No. 5,859,221 and U.S. Pat. No.5,852,188, each of which are hereby incorporated by reference herein.

The degree of interaction between two molecules that hybridize togetheris reflected by the Tm of the produced hybrid. The higher the Tm thestronger the interactions and the more stable the hybrid. Tm is effectedby numerous factors well known in the art such as the degree ofcomplementarity, the type of complementary bases present (e.g., A-Thybridization versus G-C hybridization), the structure of the nucleicacid backbones, and solution components. E.g., Sambrook et al., inMolecular Cloning, A Laboratory Manual, 2^(nd) Edition, Cold SpringHarbor Laboratory Press, 1989.

Stable hybrids are formed when the Tm of a hybrid is greater than thetemperature employed under a particular set of hybridization assayconditions. The degree of specificity of a probe can be varied byadjusting the hybridization stringency conditions. Detecting probehybridization is facilitated through the use of a detectable label.Examples of detectable labels include luminescent, enzymatic, andradioactive labels.

Examples of stringency conditions are provided in Sambrook et al., inMolecular Cloning, A Laboratory Manual, 2^(nd) Edition, Cold SpringHarbor Laboratory Press, 1989. An example of high stringency conditionsis as follows: Prehybridization of filters containing DNA is carried outfor 2 hours to overnight at 65° C. in buffer composed of 6×SSC, 5×Denhardt's solution, and 100 μg/ml denatured salmon sperm DNA. Filtersare hybridized for 12 to 48 hours at 65° C. in prehybridization mixturecontaining 100 μg/ml denatured salmon sperm DNA and 5–20×10⁶ cpm of³²P-labeled probe. Washing of filters is done at 37° C. for 1 hour in asolution containing 2×SSC, 0.1% SDS. This is followed by a wash in0.1×SSC, 0.1% SDS at 50° C. for 45 minutes before autoradiography. Otherprocedures using conditions of high stringency would include, forexample, either a hybridization step carried out in 5×SSC, 5× Denhardt'ssolution, 50% formamide at 42° C. for 12 to 48 hours or a washing stepcarried out in 0.2×SSPE, 0.2% SDS at 65° C. for 30 to 60 minutes.

Recombinant Expression

MCH receptor related polypeptides can be expressed from recombinantnucleic acid in a suitable host or in a test tube using a translationsystem. Recombinantly expressed MCH receptor polypeptides are preferablyused in assays to screen for compounds that bind to the MCH receptor andmodulate the activity of the receptor.

Preferably, expression is achieved in a host cell using an expressionvector. An expression vector contains recombinant nucleic acid encodingfor a desired polypeptide along with regulatory elements for propertranscription and processing. The regulatory elements that may bepresent include those naturally associated with the recombinant nucleicacid and exogenous regulatory elements not naturally associated with therecombinant nucleic acid. Exogenous regulatory elements such as anexogenous promoter can be useful for expressing recombinant nucleic acidin a particular host.

Generally, the regulatory elements that are present include atranscriptional promoter, a ribosome binding site, a terminator, and anoptionally present operator. Another preferred element is apolyadenylation signal providing for processing in eukaryotic cells.Preferably, an expression vector also contains an origin of replicationfor autonomous replication in a host cell, a selectable marker, alimited number of useful restriction enzyme sites, and a potential forhigh copy number. Examples of expression vectors are cloning vectors,modified cloning vectors, specifically designed plasmids and viruses.

Expression vectors that can be used to provide suitable levels ofpolypeptide expression in different hosts are well known in the art.Mammalian expression vectors well known in the art include pcDNA3(Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5(Stratagene), EBO-pSV2-neo (ATCC 37593), pBPV-1(8-2) (ATCC 37110),pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), pCI-neo (Promega)and .lambda.ZD35 (ATCC 37565). Bacterial expression vectors well knownin the art include pET11a (Novagen), lambda gt11 (Invitrogen), pcDNAII(Invitrogen), and pKK223-3 (Pharmacia). Fungal cell expression vectorswell known in the art include pYES2 (Invitrogen), Pichia expressionvector (Invitrogen). Insect cell expression vectors well known in theart include Blue Bac III (Invitrogen).

Recombinant host cells may be prokaryotic or eukaryotic. Examples ofrecombinant host cells include the following: bacteria such as E. coli;fungal cells such as yeast; mammalian cells such as human, bovine,porcine, monkey and rodent; and insect cells such as Drosophila andsilkworm derived cell lines. Commercially available mammalian cell linesinclude L cells L-M (TK.sup.-) (ATCC CCL 1.3), L cells L-M (ATCC CCL1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1(ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCCCCL 92), NTH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C1271 (ATCC CRL1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC. CCL 171).

Expression vectors may be introduced into host cells using standardtechniques. Examples of such techniques include transformation,transfection, lipofection, protoplast fusion, and electroporation.

MCH receptor nucleic acid can be expressed in a cell without the use ofan expression vector employing, for example, synthetic mRNA or nativemRNA. Additionally, mRNA can be translated in various cell-free systemssuch as wheat germ extracts and reticulocyte extracts, as well as incell based systems, such as frog oocytes. Introduction of mRNA into cellbased systems can be achieved, for example, by microinjection.

EXAMPLES

Examples are provided below to further illustrate different features ofthe present invention. The examples also illustrate useful methodologyfor practicing the invention. These examples do not limit the claimedinvention.

Example 1 Cloning of a Dog MCH Receptor

A complete coding sequence for a dog MCH receptor was obtained by RT-PCRand hybridization screening of a cDNA library (constructed in themammalian expression vector pcDNA 3.1 (+); Invitrogen) prepared from doghypothalamus poly (A)+mRNA. A forward (sense) PCR primer of SEQ. ID. NO.17 (ATG GAC CTG) derived from a human MCH receptor sequence was used toamplify the dog MCH receptor. Thus, the resulting dog MCH receptorcontains 6 amino acids (SEQ. ID. NO. 18: DLEASL) adjacent to theN-terminal methionine that may be similar or identical to those aminoacids that correspond to the naturally occurring dog MCH receptor. The Nterminal methionine (ATG) would be identical in both dog and human MCHreceptors. The remaining 353 amino acids (amino acid 8 to 353) were froma dog MCH receptor.

The dog MCH receptor protein sequence is highly related to the human andrat MCH receptor of SEQ. ID. NOs. 2 and 3. The percent protein sequenceidentity to the human and rat MCH receptors is 97.5% and 94.6%,respectively.

The amino acid and encoding nucleotide sequences for the dog MCHreceptor is as follows:

Dog MCH Receptor Amino Acid Sequence:MDLEASLLPPGPNASNTSEGPDNLTSAGPPRRTGNVSYINIIMPSVFGTICLLGII SEQ. ID. NO. 1GNSTVIFAVVKKSKLHWCSNVPDIFIINLSVVDLLFLLGMPFMIHQLMGNGVWHFGETMCTLITAMDANSQFTSTYILTAMAIDRYLATVHPISSTKFRKPSVATLVICLLWALSFISITPVWLYARLIPFPGGTVGCGIRLPNPDTDLYWFTLYQFFLAFALPFVVITAAYVRILQRMMSSVAPASQRSIRLRTKRVTRTAIAICLVFFVCWAPYYVLQLTQLSISRPTLTFVYLYNAAISLGYANSCLNPFVYIVLCETFRKRLVLSVKPAAQGQLRAVSNAQTADEERTESKGT Dog MCH Receptor Encoding NucleotideSequence (including stop codon):ATGGACCTGGAAGCCTCGCTGCTGCCCCCCGGCCCCAACGCCAGCAACAC SEQ. ID. NO. 4CTCGGAGGGCCCGGACAACCTCACCTCTGCCGGGCCACCTCGTCGCACAGGGAATGTCTCCTACATCAACATCATCATGCCTTCCGTGTTCGGCACCATCTGCCTGCTGGGTATCATCGGGAACTCCACAGTCATCTTCGCGGTGGTGAAGAAGTCCAAACTGCACTGGTGCAGCAATGTCCCCGACATCTTTATCATCAACCTCTCGGTGGTAGACCTCCTCTTTCTCCTGGGCATGCCCTTCATGATCCACCAGCTCATGGGCAATGGTGTTTGGCATTTTGGAGAGACCATGTGCACACTCATCACGGCCATGGACGCCAACAGTCAATTCACCAGCACCTACATCCTGACCGCCATGGCCATTGACCGCTACCTGGCCACTGTCCACCCCATCTCCTCCACCAAGTTCCGGAAGCCCTCTGTGGCCACCCTGGTGATCTGCCTCCTATGGGCCCTCTCATTCATCAGCATCACCCCCGTGTGGCTCTACGCTAGGCTTATCCCCTTCCCAGGGGGCACAGTGGGCTGTGGCATCCGCCTGCCCAACCCAGACACTGACCTTTACTGGTTCACCCTGTACCAGTTCTTCCTGGCCTTTGCCCTGCCCTTCGTGGTCATCACAGCCGCGTATGTGAGGATCCTGCAGCGCATGATGTCCTCGGTAGCCCCTGCCTCTCAACGCAGCATCCGGCTGCGGACAAAGAGGGTGACTCGCACGGCCATTGCCATCTGCCTGGTCTTCTTCGTGTGCTGGGCTCCCTACTATGTGCTACAGTTGACCCAGTTGTCCATCAGCCGCCCGACACTCACCTTTGTCTACCTGTACAACGCAGCCATCAGCTTGGGCTATGCCAACAGCTGCCTAAACCCCTTTGTGTACATCGTGCTCTGTGAGACATTCCGCAAGCGCTTGGTCCTGTCGGTGAAGCCTGCCGCCCAGGGGCAGCTTCGAGCCGTCAGCAATGCTCAGACAGCTGATGAGGAGAGGACAGAAAGCAAAGGCACCT GA

Example 2 Expression of Dog MCH Receptor

Measurement of MCH receptor expression in the aequorin-expressing stablereporter cell line 293-AEQ17 (Button et al., 1993, Cell Calcium, 14,663–671) was performed using a Luminoskan RT luminometer (LabsystemsInc., Gaithersburg, Md.) controlled by custom software written for aMacintosh PowerPC 6100. 293-AEQ17 cells (8×10⁵ cells plated 18 hoursbefore transfection in a T75 flask) were transfected with 22 μg of dogMCH receptor plasmid DNA: 264 μg lipofectamine.

Following approximately 40 hours of expression the apo-aequorin in thecells was charged for 4 hours with coelenterazine (10 μM) under reducingconditions (300 μM reduced glutathione) in ECB buffer (140 mM NaCl, 20mM KCl, 20 mM HEPES-NaOH [pH=7.4], 5 mM glucose, 1 mM MgCl₂, 1 mM CaCl₂,0.1 mg/ml bovine serum albumin). The cells were harvested, washed oncein ECB medium and resuspended to 500,000 cells/ml. 100 μl of cellsuspension (corresponding to 5×10⁴ cells) was then injected into thetest plate containing MCH, and the integrated light emission wasrecorded over 30 seconds, in 0.5 second units. 20 μL of lysis buffer(0.1% final Triton X-100 concentration) was then injected and theintegrated light emission recorded over 10 seconds, in 0.5 second units.

The “fractional response” values for each well were calculated by takingthe ratio of the integrated response to the initial challenge to thetotal integrated luminescence including the Triton X-100 lysis response.The EC₅₀ value for activation of the dog MCH receptor was ˜30 nM.

Other embodiments are within the following claims. While severalembodiments have been shown and described, various modifications may bemade without departing from the spirit and scope of the presentinvention.

1. A purified polypeptide comprising an amino acid region of SEQ. ID.NO. 1 selected from the group consisting of: SEQ. ID. NO: 7 LEASLLPPGP,SEQ. ID. NO. 8 SEGPDNLTSAGP, SEQ. ID. NO. 9 RRTGNVSYIN, SEQ. ID. NO. 10PFPGGTVGCG, and SEQ. ID. NO. 11 ILQRMMSSVA.


2. The polypeptide of claim 1, wherein said polypeptide comprises theamino acid sequence of SEQ. ID. NO.
 1. 3. The polypeptide of claim 2,wherein said polypeptide consists of the amino acid sequence of SEQ. ID.NO.
 1. 4. A purified nucleic acid comprising a nucleotide sequenceencoding for the polypeptide of claim
 1. 5. A purified nucleic acidcomprising a nucleotide sequence region of SEQ. ID. NO. 4 selected fromthe group consisting of: SEQ. ID. NO. 12 CCTCGGAGGGCCCGGACAACC, SEQ. ID.NO. 13 ACCTCTGCCGGGCCACCTCGT, SEQ. ID. NO. 14GCCCTTCGTGGTCATCACAGCCGCGTAT, SEQ. ID. NO. 15TGTCCTCGGTAGCCCCTGCCTCTCAA, and SEQ. ID. NO. 16 TTCGAGCCGTCAGCAATGCT.


6. The purified nucleic acid of claim 5, wherein said nucleic acidcomprises the nucleotide sequence of SEQ ID NO:
 4. 7. The purifiednucleic acid of claim 6, wherein said nucleic acid consists of thenucleotide sequence of SEQ ID NO:
 4. 8. A nucleic acid comprising arecombinant nucleotide sequence encoding for a polypeptide comprising anamino acid region of SEQ. ID. NO. 1 selected from the group consistingof: SEQ. ID. NO. 7 LEASLLPPGP, SEQ. ID. NO. 8 SEGPDNLTSAGP, SEQ. ID. NO.9 RRTGNVSYIN, SEQ. ID. NO. 10 PFPGGTVGCG, and SEQ. ID. NO. 11ILQRMMSSVA.


9. The nucleic acid of claim 8, wherein said polypeptide comprises theamino acid sequence of SEQ ID NO:1.
 10. The nucleic acid of claim 8,wherein said polypeptide consists of the amino acid sequence of SEQ IDNO:1.
 11. The nucleic acid of claim 8, wherein said nucleic acid is anexpression vector.
 12. A recombinant cell comprising the expressionvector of claim
 11. 13. A recombinant cell made by a process comprisingthe step of introducing into said cell the expression vector of claim11.
 14. A method of measuring the ability of a test compound to affectMCH receptor activity comprising the steps of: a) contacting arecombinant cell with said compound, wherein said recombinant cellcomprises a recombinant nucleic acid expressing a functional MCHreceptor that comprises the amino acid sequence of SEQ. ID. NO. 1; andb) measuring MCH receptor activity.
 15. The method of claim 14, whereinsaid MCH receptor consists of the sequence of SEQ. ID. NO.
 1. 16. Themethod of claim 15, wherein said contacting further comprises contactingthe cell with a MCH agonist to stimulate MCH receptor activity, andmeasuring the ability of said compound to modulate MCH receptoractivity.